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Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major)
Journal Pre-proof Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major) Seong Don Hwang, Kwang-Min Choi, Jee Youn Hwang, Mun-Gyeong Kwon, Ji-Min Jeong, Jung Soo Seo, Bo-Yeong Jee, Chan-Il Park PII:
S1050-4648(19)31229-X
DOI:
https://doi.org/10.1016/j.fsi.2019.12.090
Reference:
YFSIM 6732
To appear in:
Fish and Shellfish Immunology
Received Date: 17 October 2019 Revised Date:
25 December 2019
Accepted Date: 28 December 2019
Please cite this article as: Hwang SD, Choi K-M, Hwang JY, Kwon M-G, Jeong J-M, Seo JS, Jee BY, Park C-I, Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major), Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/ j.fsi.2019.12.090. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Molecular genetic characterisation and expression profiling of calpain 3 transcripts in
2
red sea bream (Pagrus major)
3 4 5 6 7 8 9
Seong Don Hwang 1, a, Kwang-Min Choi 1, b, Jee Youn Hwang a, Mun-Gyeong Kwon a, Ji-Min Jeong a, Jung Soo Seo a, Bo-Yeong Jee a, Chan-Il Park b * a Aquatic Animal Disease Control Center, National Institute of Fisheries Science (NIFS), 216 Gijanghaean-ro, Gijang-eup, Gijang-gun, Busan 46083, Republic of Korea b Institute of Marine Industry, College of Marine Science, Gyeongsang National University, 455, Tongyeong 650-160, Republic of Korea
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1
These authors contributed equally to this work.
Contact email: [email protected] (C.-I. Park)
27 28 1
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Abstract
30
Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are
31
calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological
32
processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus
33
major (PmCAPN3) and confirmed the conserved molecular biological properties in the
34
predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3
35
were found to be highly conserved in all of the examined species, and one catalytic domain
36
and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA
37
was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the
38
other tissues used in the experiment. After artificial infections with fish pathogens, significant
39
changes in its expression levels were found in immune-related tissues, most of showed
40
upregulation. In particular, the highest level of expression was found in the liver, a tissue
41
associated with protease activity. Taken together, these results suggest a physiological
42
activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been
43
reported in the immune system.
44 45 46 47 48 49 50 51 52
Key words: Pagrus major, Streptococcus iniae, Edwardsiella piscicida, Red sea bream
53
iridovirus, Calpain 3 2
54
1. Introduction
55
Proteases play essential roles in basic biological processes of hosts and pathogens, among
56
which cysteine proteases play roles in the recognition, elimination, signalling and cellular
57
homeostasis related to antigens and pathogens in both the innate and adaptive immune
58
responses [1]. Calpain (CAPN) is a calcium-dependent cytoplasmic cysteine protease that
59
belongs to the papain superfamily, and it has been identified in most eukaryotes and some
60
bacteria. These families are reported to have 15 isoforms in humans and mammals, and the
61
proteins are involved in the regulation of numerous physiological processes including cell
62
proliferation, apoptosis, membrane fusion, cell motility, signal transduction and platelet
63
activation [2]. Increased intracellular calcium level activates CAPN, which then induces cell
64
death via destruction of the cell membrane structure, cytoplasm and nuclear matrix; therefore,
65
their activity is extensively regulated [3]. The CAPN family can be classified into ubiquitous
66
and tissue-specific types according to their expression characteristics [4].
67
CAPN3 (also known as p94) is a specific CAPN family that is specifically expressed in
68
muscle tissue, and it is mostly present in muscle in an inactive state. Mutations or defects can
69
occur that lead to severe muscle loss or impairment [5]. CAPN3 was first detected in 1989 in
70
rat cDNA samples, and it has since been reported mostly in vertebrates. In addition, CAPN3
71
is a protein and non-proteolytic enzyme that is known to be the only enzyme activated by
72
sodium ions. Since CAPN3 is expressed in immune cells in addition to muscle tissue, loss of
73
CAPN3 function can cause immunological defects [6]. Unique and specific studies of CAPN3,
74
as described above, are still lacking in teleosts, and there are no reports of immunological
75
studies.
76
Red sea bream (Pagrus major) is a very popular fish species in South Korea and Japan,
77
and it is relevant in various contexts, including leisure fishing and as food. The animals are
78
mainly cultured in cages, and aquaculture in marine cage facilities is difficult to manage due 3
79
to environmental factors and diseases. P. major is vulnerable to bacterial and viral diseases
80
that can lead to mass mortality, especially infections by red sea bream iridovirus (RSIV),
81
which is prevalent in aquaculture environments [7]. Therefore, to systematically manage P.
82
major, which is an economically large important part of fisheries, it is necessary to solve the
83
problem of disease.
84
In this study, we obtained the cDNA sequence of PmCAPN3 from P. major and analysed
85
its molecular characteristics and mRNA expression pattern. As a result, we suggest that
86
PmCAPN3 has a possible immunological function in P. major.
87 88
2. Materials and methods
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2.1. Fish and pathogenic microorganism
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Healthy P. major (weight: 173.2 ± 31.1 g, body length: 22.4 ± 0.9 cm) was provided by the
91
Gyeongsangnam-do Fisheries Resources Research Institute, and the animals were kept in the
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aquarium for two weeks. During the acclimatization period, the water temperature was
93
maintained at 21 ± 1 °C, with constant aeration and salinity, and feeding was carried out
94
twice a day.
95
For the pathogen challenge tests, the pathogenic bacteria and RSIV used in artificial
96
infection experiments for the gene expression analysis were provided by the Fish Pathology
97
Division of the National Institute of Fisheries Science (Busan, Republic of Korea). The
98
strains were Streptococcus iniae (S. iniae) FP5228 and Edwardsiella piscicida (E. piscicida)
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FSW910410. The bacteria were cultured in brain heart infusion broth (BD Difco, USA) at
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28 °C.
101 102 103
2.2. Gene cloning and bioinformatic analyses The open reading frame (ORF) cDNA sequence of PmCAPN3 was obtained based on 4
104
previously collected genome/transcript next generation sequencing (NGS) data [8]. The
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integrity of the cDNA sequence was confirmed via Sanger sequencing after cloning and via
106
prediction of the amino acid sequence. Based on the amino acid sequence, characteristic
107
domains were predicted using the Expert Protein Analysis System PROSITE Scan tool
108
(http://prosite.expasy.org). Furthermore, the predicted amino acid sequence of PmCAPN3
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was confirmed to contain the relevant sequence using the BLAST algorithm of the National
110
Biotechnology Information Center (http://www.ncbi.nlm.nih.gov/blast). A multiple sequence
111
alignment analysis with related CAPN3 amino acid sequences was performed with ClustalX
112
version 2.1, and the results were visualized with GeneDoc version 2.7. A phylogenetic
113
reconstruction was generated using the neighbour-joining method with a bootstrap of 1,000
114
replicate datasets implemented in the Molecular Evolutionary Genetics Analysis (MEGA)
115
version 6.0.
116 117
2.3. Gene expression analysis using quantitative real-time PCR (RT-qPCR)
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The expression pattern of PmCAPN3 mRNA in P. major was examined in healthy and
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artificially infected animals. To analyse the expression of PmCAPN3 in healthy animals,
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various tissue types (brain, gills, head kidney, heart, intestine, liver, muscle, skin, spleen,
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stomach and trunk kidney) were aseptically extracted from three individual fish that were
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euthanized with the anaesthetic agent benzocaine (Sigma-Aldrich, USA). Total RNA
123
extraction was performed using a TRIzol-based reagent (RNAiso Plus, Takara, Japan)
124
according to the manufacturer's manual, and genomic DNA was removed using recombinant
125
DNase I (Takara). The concentration and purity of the extracted total RNA was confirmed
126
using a NanoVue (GE Healthcare, UK) spectrophotometer, and the PrimeScriptTM 1st strand
127
cDNA Synthesis Kit (Takara) was used to synthesize the first-strand cDNA from the mRNA
128
template according to the manufacturer's manual. The synthesized cDNA was analysed via 5
129
quantitative real-time PCR (RT-qPCR) using TB GreenTM Premix Ex TaqTM (Takara) and
130
specific primers (forward: 5′-GAAGAGGATGACGACCCAGA-3′ and reverse: 5′-
131
TGCTGGTTCTGTCCACACAT-3′) to measure the expression levels of PmCAPN3 mRNA.
132
The cycle threshold (Ct) values were normalized using elongation factor 1 alpha of P. major
133
(PmEF-1α), and the relative expression levels were calculated via the delta-delta Ct method.
134
The results are expressed as the fold changes relative to the reference value (stomach). The
135
primer
136
CCTTCAAGTACGCCTGGGTG-3′ and reverse: 5′- CTGTGTCCAGGGGCATCAAT-3′.
sequences
for
PmEF-1α
were
as
follows:
forward:
5′-
137
To examine the expression of the PmCAPN3 gene upon pathogenic infection, healthy fish
138
were artificially infected with bacteria or virus, and the level of PmCAPN3 mRNA was
139
measured via RT-qPCR. First, the fish were divided into three experimental groups and
140
injected with S. iniae (1 × 105 CFU/fish), E. piscicida (1 × 105 CFU/fish) or RSIV (1 × 106
141
copies/fish), and the water temperature was maintained at 23 ± 1 ℃. Three fish were
142
randomly selected 0, 1 and 12 hours post-infection (hpi) and 1, 3, 5 and 7 days post-infection
143
(dpi) and euthanized via anaesthesia. Next, samples of four tissue types (gills, whole kidney,
144
liver and spleen) were aseptically extracted. Total RNA extraction, cDNA synthesis and RT-
145
qPCR were conducted as described above.
146 147
2.4. Statistical data analysis
148
All samples were analysed in triplicate, and the results are reported as the mean ± standard
149
deviation (SD). The statistical analysis was performed using SPSS software version 19 (IBM,
150
USA). The PmCAPN3 expression levels in tissues during pathogen infection were assessed
151
via one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.
152 153
3. Results and discussion 6
154
3.1. Gene structure and sequence features of the PmCAPN3 amino acid sequence
155
CAPN is a cytoplasmic protease that exhibits calcium-dependent proteolytic activity at the
156
neutral pH the cell, and a wide range of biological functions have been reported based on its
157
selective cleavage activity, which is tightly regulated by an endogenous inhibitor [9–11]. We
158
examined the molecular and gene expression characteristics after obtaining the PmCAPN3
159
sequence from P. major. A multiple sequence alignment of PmCAPN3 (accession number:
160
MN105088) and CAPN3 amino acid sequences from other species in the NCBI database
161
showed that the N-terminal catalytic domain (77 to 374 aa) and four EF-hand calcium
162
binding sites (615 to 633, 642 to 677, 680 to 707 and 737 to 771 aa) were highly conserved.
163
CAPN3 has a relatively low calcium requirement, but it was fully activated by calcium even
164
after experimental mutation of its proteolytic core [12,13]. In addition, it was reported that
165
another classic CAPN could compensate when a serious defect occurred in CAPN3 [6].
166
However, genetic defects in CAPN are of great biological importance, as they can cause
167
lethality and functional defects [14,15]. The homology between the sequences of the
168
compared species showed relatively high similarities, ranging from 64.9 to 89.9% (Fig. 1).
169
The highest homology was with Atlantic halibut CAPN3 (89.9%) followed by turbot (89.2%).
170
The CAPN amino acid sequences previously reported in other vertebrate species were highly
171
conserved, with at least 90% homology [11]. Although these results and previous reports
172
confirm that PmCAPN3 has been highly conserved, it is thought that it can be functionally
173
maintained or replaced in the event of defects or mutations.
174
Based on a phylogenetic analysis constructed via the neighbour-joining method, the
175
PmCAPN3 sequence belongs to the CAPN3 group with a bootstrap value of 100% (Fig. 2).
176
PmCAPN3 was also included in the teleost cluster, and the closest flexible relationship with
177
CAPN3 in marine fish was identified.
178 7
179
3.2. In vivo expression patterns of PmCAPN3 in various tissues
180
CAPNs are widely present in a variety of organisms from humans to microorganisms, and
181
CAPN3 mRNA is known to be present in most tissue types [16]. Previous studies have
182
reported that CAPN3 is the only muscle-specific CAPN member, and it is much more
183
strongly expressed in skeletal muscle, especially fast (type II) fibres, than in other tissues. It
184
is also known to be expressed in a variety of tissues; therefore, it does not show tissue-
185
specific expression. Some reports have shown that, it is more abundant in the skin than in
186
muscle in humans [17,18]. In healthy P. major, PmCAPN3 mRNA was ubiquitously
187
distributed and relatively abundant in muscle and skin (1199- and 220-fold, respectively) (Fig.
188
3). In addition, relatively high levels were expressed in the spleen and kidney, the major
189
immune-related tissues of bony fish [19]. CAPN3 transcripts are expressed not only in the
190
skeletal muscle of chicken but also in the brain, liver and heart, and CAPN3 mRNA has been
191
detected in the heart of human embryos, although no protein was detected [20,21]. In
192
zebrafish, two CAPN3s showed distinct differences in their expression patterns during early
193
development; CAPN3a was abundant in the eye and brain while and CAPN3b was abundant
194
in the digestive organs and head regions [22]. These results indicate that the tissue-specific
195
distribution of CAPN3 varies according to evolutionary or developmental characteristics, and
196
PmCAPN3 transcripts are expressed in various immune-related tissues types in addition to
197
muscle and skin; thus, PmCAPN3 expression might be related to various immunological or
198
biological functions.
199 200
3.3. In vivo expression pattern of PmCAPN3 upon pathogen challenge
201
CAPN is known to be a disease-causing enzyme based on some reports, and its activation
202
can exacerbate pathological conditions; however, CAPN3 is generally reported to have
203
protective functions against disease [6,23–25]. Therefore, we evaluated mRNA expression 8
204
levels in immune-related organs (the gills, kidneys, liver, and spleen) of pathogen-challenged
205
fish to determine the expression levels of PmCAPN3 in the immune system of P. major. Total
206
RNA was extracted from various tissue samples and examined via RT-qPCR. After S. iniae
207
challenge, the PmCAPN3 mRNA was upregulated in all of the examined tissue types (Fig. 4).
208
Its expression reached the highest level 1 dpi and then decreased below the baseline level.
209
Importantly, the expression level of the PmCAPN3 transcript was significantly highest 12 hpi
210
in the kidney, and its expression level was maintained at a significantly higher level after 1
211
dpi. After E. piscicida challenge, the PmCAPN3 mRNA level was significantly upregulated
212
in the kidneys, liver and spleen, while its expression levels in the gills were significantly
213
downregulated during the infection period. The expression levels of the PmCAPN3 transcript
214
in the kidneys and liver were significantly higher 3 dpi but decreased 5 dpi after the largest
215
increase occurred in the spleen 1 dpi. After RSIV challenge, significantly higher upregulation
216
was observed in the gills, kidneys and liver 3 dpi, but the expression level was downregulated
217
or maintained at the baseline level in the spleen. Tissue-specific expression analysis revealed
218
that the mRNA levels of PmCAPN3, a member of the cysteine protein family C2, were
219
significantly higher in the liver after pathogen infection than in other immune-related organs.
220
In previous reports, the cysteine proteinase family C1 (cathepsin B and L) was upregulated in
221
the liver following fungal and bacterial infections [26,27]. The liver plays an important role
222
in the innate immune response and is a key tissue for protein synthesis, which is essential for
223
protein homeostasis [28,29]. These results suggest that expression of the PmCAPN3
224
transcripts is induced and that the protein is activated mainly in the liver during pathogen
225
infection. CAPN3 was highly expressed in resistant animals [30]; however, our study also
226
showed significant increases in expression in immune-related tissues of P. major following
227
artificial infection with low concentrations of pathogens. The activity of CAPN protein is
228
increased in Leo virus-infected muscle cells, which is associated with apoptotic cell death 9
229
induced by viral diseases [31]. On the other hand, calpain-inhibited rats showed a significant
230
reduction in mortality after artificial infection with influenza A virus [32]. These results
231
suggest that it is important to control or understand CAPN signalling in diseases caused by
232
viral invasion. CAPN3 lacking protease activity was detected at high levels in human
233
melanoma cell lines, and CAPN3 is known to act as a regulator of existing CAPN systems
234
[33,34]. Furthermore, the CAPN3 gene is thought to be an important molecular marker in
235
aquaculture because it has been identified as a major muscle growth factor in breeding studies
236
of poultry and cattle [35–38]. Therefore, future studies on P. major breeding might suggest
237
that PmCAPN3 could be used as a candidate molecular marker. On the other hand, it was
238
significantly downregulated in the gills during nearly all periods of artificial infection by E.
239
piscicida (Fig. 4B). Previous studies have shown that E. tarda infects fish through the gills, a
240
major entry point [39] and organ vulnerable to infection by E. tarda. The infection is thought
241
to cause significant damage to the gills. Similar results confirmed that PmCAPN3 mRNA was
242
downregulated in the spleen, the organ most sensitive to RSIV infection (Fig. 4C). These
243
events might have disrupted the immune system in the tissues of hosts vulnerable to pathogen
244
invasion.
245
Our results also indicate that the PmCAPN3 mRNA expression level was upregulated by
246
pathogen infection via an in vivo analysis; however, this immunological result is not clear as
247
the protein level was not confirmed. CAPN3 is difficult to successfully purify because of its
248
strong and rapid autolysis, and full-length wild-type protein is a very unstable, making it
249
difficult to manipulate in vitro. Moreover, this protein shows rapid autolysis even in sodium-
250
containing solutions, such as buffered saline; thus, relevant studies are lacking, especially in
251
bony fish. This study identified significant upregulation of the PmCAPN3 transcript level
252
following pathogen invasion, confirming its potential as a pathogenic disease marker;
253
however, it also identified the need for further studies at the protein level. 10
254 255
Acknowledgements
256
This work was supported by a grant from the National Institute of Fisheries Science
257
(R2019058) and 'Smart Aquaculture Research Center', funded by the Ministry of Oceans and
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Fisheries, Korea.
259 260
261
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32–39.
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Calpain 3 gene is associated with meat tenderness in zebu and composite breeds of
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cattle. BMC Genet. 9, 41.
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Identification and association of the single nucleotide polymorphisms in calpain3
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(CAPN3) gene with carcass traits in chickens, BMC Genet. 10, 10.
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A.M., Felício, C., Boschiero, J.C., Balieiro, M.C., Ledur, J.B., Ferraz, T., Michelan
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Filho, A.S., Moura, L.L., Coutinho, 2013. Identification and association of
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polymorphisms in CAPN1 and CAPN3 candidate genes related to performance and
374
meat quality traits in chickens, Genet. Mol. Res. 12, 472–482. 15
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H., Xu, Y., Zhang, Q., Zou, L., Li, C., Han, H., Liu, J., Hu, T., Zhong, Y., Wang,
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Muscle Tissues of Sichuan White Geese at Different Growth Stages, The Journal of
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379
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S.H., Ling, X.H., Wang, L., Xie, T.M., Lim, K.Y., Leung, 2000. Use of green
380
fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in
381
vivo and in vitro fish models, Microbiology 146, 7–19.
382 383
Figure legends
384
Figure 1. Multiple alignment analysis of the deduced amino acid sequences of PmCAPN3
385
and other known CAPN sequences. This analysis is based on the following sequence data:
386
Atlantic halibut (ACY78226), turbot (AWP20177), Atlantic salmon (ACN10671), house
387
mouse (AAD28255) and human (AAD28253). The predicted catalytic domain and four EF-
388
hand calcium binding sites are indicated by the arrows and boxes, respectively. Black boxes:
389
identity = 100%; Grey boxes: 80% ≤ identity < 100%; Light grey boxes: 50% ≤ identity <
390
80%.
391
Figure 2. A phylogenetic tree based on the neighbour-joining method of PmCAPN and other
392
known CAPN homologues. The scale bar indicates a branch length of 0.2. The numbers show
393
the bootstrap percentiles from 1,000 replicates. The GenBank accession numbers are as
394
follows: Atlantic halibut CAPN3 (ACY78226), turbot CAPN3 (AWP20177), Atlantic salmon
395
CAPN3 (ACN10671), house mouse CAPN3 (AAD28255), human CAPN3 (AAD28253),
396
house mouse CAPN1 (AAH26138), house mouse CAPN2 (AAH54726), human CAPN5
397
(AAH18123), human CAPN6 (AAH00730), human CAPN7 (AAH56202), human CAPN8
398
(AAI57894), human CAPN9 (AAW49735), human CAPN10 (AAH07553), human CAPN11 16
399
(AAH33733), human CAPN12 (NP_653292), human CAPN13 (AAI17346), cattle CAPN14
400
(DAA24548) and human CAPN15 (NP_005623).
401
Figure 3. RT-qPCR-based analysis of the PmCAPN3 mRNA expression levels in various
402
tissue types in healthy Pagrus major. The PmEF-1α gene was used to normalise the RT-
403
qPCR results. The expression levels are reported as fold increases relative to the value in the
404
stomach. All data are presented as the mean ± SD from five independent cDNA samples with
405
three replicates per sample (N = 3).
406
Figure 4. RT-qPCR-based analysis of the PmCAPN3 mRNA levels in the gills, kidneys, liver
407
and spleen of Pagrus major after infection with (A) Streptococcus iniae, (B) Edwardsiella
408
piscicida or (C) red sea bream iridovirus (RSIV). The PmCAPN3 levels were quantified
409
relative to that of the PmEF-1α gene. The gene expression levels are represented as the
410
mean ± SD (N = 3). Asterisks indicate significant differences (*P < 0.05, **P < 0.01) versus
411
the control (0 h).
17
Catalytic domain Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Figure 1. Hwang et al.
●
Figure 2. Hwang et al.
Figure 3. Hwang et al.
12
Relative expression level (Fold)
(A) S. iniae
**
10 8 6
**
**
4
*
*
* **
* *
2 *
0
Gill Relative expression level (Fold)
(B) E. piscicida
Kidney
Liver
Spleen
8
**
6
Cont. (0 h) 1h 12 h 1d 3d 5d 7d
** **
4
** *
*
2 ** ** ** ** **
*
*
0
Gill Relative expression level (Fold)
(C) RSIV
Kidney
Liver
25
Spleen
**
20 15 *
10
**
5
** ** *
*
0
Gill
Kidney
Liver
Figure 4. Hwang et al.
Spleen
Highlight The calpain 3 (CAPN3) was identified by NGS analysis from red sea bream (Pagrus major). PmCAPN3 showed high sequence similarity with other species CAPN3. PmCAPN3 mRNA was most abundantly distributed in the muscle and skin of healthy P. major. PmCAPN3 mRNA by fish pathogen challenge has showed a significant increase or decrease, and particularly showed the highest expression level in the liver.
S1050-4648(19)31229-X
DOI:
https://doi.org/10.1016/j.fsi.2019.12.090
Reference:
YFSIM 6732
To appear in:
Fish and Shellfish Immunology
Received Date: 17 October 2019 Revised Date:
25 December 2019
Accepted Date: 28 December 2019
Please cite this article as: Hwang SD, Choi K-M, Hwang JY, Kwon M-G, Jeong J-M, Seo JS, Jee BY, Park C-I, Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major), Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/ j.fsi.2019.12.090. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Molecular genetic characterisation and expression profiling of calpain 3 transcripts in
2
red sea bream (Pagrus major)
3 4 5 6 7 8 9
Seong Don Hwang 1, a, Kwang-Min Choi 1, b, Jee Youn Hwang a, Mun-Gyeong Kwon a, Ji-Min Jeong a, Jung Soo Seo a, Bo-Yeong Jee a, Chan-Il Park b * a Aquatic Animal Disease Control Center, National Institute of Fisheries Science (NIFS), 216 Gijanghaean-ro, Gijang-eup, Gijang-gun, Busan 46083, Republic of Korea b Institute of Marine Industry, College of Marine Science, Gyeongsang National University, 455, Tongyeong 650-160, Republic of Korea
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1
These authors contributed equally to this work.
Contact email: [email protected] (C.-I. Park)
27 28 1
29
Abstract
30
Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are
31
calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological
32
processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus
33
major (PmCAPN3) and confirmed the conserved molecular biological properties in the
34
predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3
35
were found to be highly conserved in all of the examined species, and one catalytic domain
36
and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA
37
was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the
38
other tissues used in the experiment. After artificial infections with fish pathogens, significant
39
changes in its expression levels were found in immune-related tissues, most of showed
40
upregulation. In particular, the highest level of expression was found in the liver, a tissue
41
associated with protease activity. Taken together, these results suggest a physiological
42
activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been
43
reported in the immune system.
44 45 46 47 48 49 50 51 52
Key words: Pagrus major, Streptococcus iniae, Edwardsiella piscicida, Red sea bream
53
iridovirus, Calpain 3 2
54
1. Introduction
55
Proteases play essential roles in basic biological processes of hosts and pathogens, among
56
which cysteine proteases play roles in the recognition, elimination, signalling and cellular
57
homeostasis related to antigens and pathogens in both the innate and adaptive immune
58
responses [1]. Calpain (CAPN) is a calcium-dependent cytoplasmic cysteine protease that
59
belongs to the papain superfamily, and it has been identified in most eukaryotes and some
60
bacteria. These families are reported to have 15 isoforms in humans and mammals, and the
61
proteins are involved in the regulation of numerous physiological processes including cell
62
proliferation, apoptosis, membrane fusion, cell motility, signal transduction and platelet
63
activation [2]. Increased intracellular calcium level activates CAPN, which then induces cell
64
death via destruction of the cell membrane structure, cytoplasm and nuclear matrix; therefore,
65
their activity is extensively regulated [3]. The CAPN family can be classified into ubiquitous
66
and tissue-specific types according to their expression characteristics [4].
67
CAPN3 (also known as p94) is a specific CAPN family that is specifically expressed in
68
muscle tissue, and it is mostly present in muscle in an inactive state. Mutations or defects can
69
occur that lead to severe muscle loss or impairment [5]. CAPN3 was first detected in 1989 in
70
rat cDNA samples, and it has since been reported mostly in vertebrates. In addition, CAPN3
71
is a protein and non-proteolytic enzyme that is known to be the only enzyme activated by
72
sodium ions. Since CAPN3 is expressed in immune cells in addition to muscle tissue, loss of
73
CAPN3 function can cause immunological defects [6]. Unique and specific studies of CAPN3,
74
as described above, are still lacking in teleosts, and there are no reports of immunological
75
studies.
76
Red sea bream (Pagrus major) is a very popular fish species in South Korea and Japan,
77
and it is relevant in various contexts, including leisure fishing and as food. The animals are
78
mainly cultured in cages, and aquaculture in marine cage facilities is difficult to manage due 3
79
to environmental factors and diseases. P. major is vulnerable to bacterial and viral diseases
80
that can lead to mass mortality, especially infections by red sea bream iridovirus (RSIV),
81
which is prevalent in aquaculture environments [7]. Therefore, to systematically manage P.
82
major, which is an economically large important part of fisheries, it is necessary to solve the
83
problem of disease.
84
In this study, we obtained the cDNA sequence of PmCAPN3 from P. major and analysed
85
its molecular characteristics and mRNA expression pattern. As a result, we suggest that
86
PmCAPN3 has a possible immunological function in P. major.
87 88
2. Materials and methods
89
2.1. Fish and pathogenic microorganism
90
Healthy P. major (weight: 173.2 ± 31.1 g, body length: 22.4 ± 0.9 cm) was provided by the
91
Gyeongsangnam-do Fisheries Resources Research Institute, and the animals were kept in the
92
aquarium for two weeks. During the acclimatization period, the water temperature was
93
maintained at 21 ± 1 °C, with constant aeration and salinity, and feeding was carried out
94
twice a day.
95
For the pathogen challenge tests, the pathogenic bacteria and RSIV used in artificial
96
infection experiments for the gene expression analysis were provided by the Fish Pathology
97
Division of the National Institute of Fisheries Science (Busan, Republic of Korea). The
98
strains were Streptococcus iniae (S. iniae) FP5228 and Edwardsiella piscicida (E. piscicida)
99
FSW910410. The bacteria were cultured in brain heart infusion broth (BD Difco, USA) at
100
28 °C.
101 102 103
2.2. Gene cloning and bioinformatic analyses The open reading frame (ORF) cDNA sequence of PmCAPN3 was obtained based on 4
104
previously collected genome/transcript next generation sequencing (NGS) data [8]. The
105
integrity of the cDNA sequence was confirmed via Sanger sequencing after cloning and via
106
prediction of the amino acid sequence. Based on the amino acid sequence, characteristic
107
domains were predicted using the Expert Protein Analysis System PROSITE Scan tool
108
(http://prosite.expasy.org). Furthermore, the predicted amino acid sequence of PmCAPN3
109
was confirmed to contain the relevant sequence using the BLAST algorithm of the National
110
Biotechnology Information Center (http://www.ncbi.nlm.nih.gov/blast). A multiple sequence
111
alignment analysis with related CAPN3 amino acid sequences was performed with ClustalX
112
version 2.1, and the results were visualized with GeneDoc version 2.7. A phylogenetic
113
reconstruction was generated using the neighbour-joining method with a bootstrap of 1,000
114
replicate datasets implemented in the Molecular Evolutionary Genetics Analysis (MEGA)
115
version 6.0.
116 117
2.3. Gene expression analysis using quantitative real-time PCR (RT-qPCR)
118
The expression pattern of PmCAPN3 mRNA in P. major was examined in healthy and
119
artificially infected animals. To analyse the expression of PmCAPN3 in healthy animals,
120
various tissue types (brain, gills, head kidney, heart, intestine, liver, muscle, skin, spleen,
121
stomach and trunk kidney) were aseptically extracted from three individual fish that were
122
euthanized with the anaesthetic agent benzocaine (Sigma-Aldrich, USA). Total RNA
123
extraction was performed using a TRIzol-based reagent (RNAiso Plus, Takara, Japan)
124
according to the manufacturer's manual, and genomic DNA was removed using recombinant
125
DNase I (Takara). The concentration and purity of the extracted total RNA was confirmed
126
using a NanoVue (GE Healthcare, UK) spectrophotometer, and the PrimeScriptTM 1st strand
127
cDNA Synthesis Kit (Takara) was used to synthesize the first-strand cDNA from the mRNA
128
template according to the manufacturer's manual. The synthesized cDNA was analysed via 5
129
quantitative real-time PCR (RT-qPCR) using TB GreenTM Premix Ex TaqTM (Takara) and
130
specific primers (forward: 5′-GAAGAGGATGACGACCCAGA-3′ and reverse: 5′-
131
TGCTGGTTCTGTCCACACAT-3′) to measure the expression levels of PmCAPN3 mRNA.
132
The cycle threshold (Ct) values were normalized using elongation factor 1 alpha of P. major
133
(PmEF-1α), and the relative expression levels were calculated via the delta-delta Ct method.
134
The results are expressed as the fold changes relative to the reference value (stomach). The
135
primer
136
CCTTCAAGTACGCCTGGGTG-3′ and reverse: 5′- CTGTGTCCAGGGGCATCAAT-3′.
sequences
for
PmEF-1α
were
as
follows:
forward:
5′-
137
To examine the expression of the PmCAPN3 gene upon pathogenic infection, healthy fish
138
were artificially infected with bacteria or virus, and the level of PmCAPN3 mRNA was
139
measured via RT-qPCR. First, the fish were divided into three experimental groups and
140
injected with S. iniae (1 × 105 CFU/fish), E. piscicida (1 × 105 CFU/fish) or RSIV (1 × 106
141
copies/fish), and the water temperature was maintained at 23 ± 1 ℃. Three fish were
142
randomly selected 0, 1 and 12 hours post-infection (hpi) and 1, 3, 5 and 7 days post-infection
143
(dpi) and euthanized via anaesthesia. Next, samples of four tissue types (gills, whole kidney,
144
liver and spleen) were aseptically extracted. Total RNA extraction, cDNA synthesis and RT-
145
qPCR were conducted as described above.
146 147
2.4. Statistical data analysis
148
All samples were analysed in triplicate, and the results are reported as the mean ± standard
149
deviation (SD). The statistical analysis was performed using SPSS software version 19 (IBM,
150
USA). The PmCAPN3 expression levels in tissues during pathogen infection were assessed
151
via one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.
152 153
3. Results and discussion 6
154
3.1. Gene structure and sequence features of the PmCAPN3 amino acid sequence
155
CAPN is a cytoplasmic protease that exhibits calcium-dependent proteolytic activity at the
156
neutral pH the cell, and a wide range of biological functions have been reported based on its
157
selective cleavage activity, which is tightly regulated by an endogenous inhibitor [9–11]. We
158
examined the molecular and gene expression characteristics after obtaining the PmCAPN3
159
sequence from P. major. A multiple sequence alignment of PmCAPN3 (accession number:
160
MN105088) and CAPN3 amino acid sequences from other species in the NCBI database
161
showed that the N-terminal catalytic domain (77 to 374 aa) and four EF-hand calcium
162
binding sites (615 to 633, 642 to 677, 680 to 707 and 737 to 771 aa) were highly conserved.
163
CAPN3 has a relatively low calcium requirement, but it was fully activated by calcium even
164
after experimental mutation of its proteolytic core [12,13]. In addition, it was reported that
165
another classic CAPN could compensate when a serious defect occurred in CAPN3 [6].
166
However, genetic defects in CAPN are of great biological importance, as they can cause
167
lethality and functional defects [14,15]. The homology between the sequences of the
168
compared species showed relatively high similarities, ranging from 64.9 to 89.9% (Fig. 1).
169
The highest homology was with Atlantic halibut CAPN3 (89.9%) followed by turbot (89.2%).
170
The CAPN amino acid sequences previously reported in other vertebrate species were highly
171
conserved, with at least 90% homology [11]. Although these results and previous reports
172
confirm that PmCAPN3 has been highly conserved, it is thought that it can be functionally
173
maintained or replaced in the event of defects or mutations.
174
Based on a phylogenetic analysis constructed via the neighbour-joining method, the
175
PmCAPN3 sequence belongs to the CAPN3 group with a bootstrap value of 100% (Fig. 2).
176
PmCAPN3 was also included in the teleost cluster, and the closest flexible relationship with
177
CAPN3 in marine fish was identified.
178 7
179
3.2. In vivo expression patterns of PmCAPN3 in various tissues
180
CAPNs are widely present in a variety of organisms from humans to microorganisms, and
181
CAPN3 mRNA is known to be present in most tissue types [16]. Previous studies have
182
reported that CAPN3 is the only muscle-specific CAPN member, and it is much more
183
strongly expressed in skeletal muscle, especially fast (type II) fibres, than in other tissues. It
184
is also known to be expressed in a variety of tissues; therefore, it does not show tissue-
185
specific expression. Some reports have shown that, it is more abundant in the skin than in
186
muscle in humans [17,18]. In healthy P. major, PmCAPN3 mRNA was ubiquitously
187
distributed and relatively abundant in muscle and skin (1199- and 220-fold, respectively) (Fig.
188
3). In addition, relatively high levels were expressed in the spleen and kidney, the major
189
immune-related tissues of bony fish [19]. CAPN3 transcripts are expressed not only in the
190
skeletal muscle of chicken but also in the brain, liver and heart, and CAPN3 mRNA has been
191
detected in the heart of human embryos, although no protein was detected [20,21]. In
192
zebrafish, two CAPN3s showed distinct differences in their expression patterns during early
193
development; CAPN3a was abundant in the eye and brain while and CAPN3b was abundant
194
in the digestive organs and head regions [22]. These results indicate that the tissue-specific
195
distribution of CAPN3 varies according to evolutionary or developmental characteristics, and
196
PmCAPN3 transcripts are expressed in various immune-related tissues types in addition to
197
muscle and skin; thus, PmCAPN3 expression might be related to various immunological or
198
biological functions.
199 200
3.3. In vivo expression pattern of PmCAPN3 upon pathogen challenge
201
CAPN is known to be a disease-causing enzyme based on some reports, and its activation
202
can exacerbate pathological conditions; however, CAPN3 is generally reported to have
203
protective functions against disease [6,23–25]. Therefore, we evaluated mRNA expression 8
204
levels in immune-related organs (the gills, kidneys, liver, and spleen) of pathogen-challenged
205
fish to determine the expression levels of PmCAPN3 in the immune system of P. major. Total
206
RNA was extracted from various tissue samples and examined via RT-qPCR. After S. iniae
207
challenge, the PmCAPN3 mRNA was upregulated in all of the examined tissue types (Fig. 4).
208
Its expression reached the highest level 1 dpi and then decreased below the baseline level.
209
Importantly, the expression level of the PmCAPN3 transcript was significantly highest 12 hpi
210
in the kidney, and its expression level was maintained at a significantly higher level after 1
211
dpi. After E. piscicida challenge, the PmCAPN3 mRNA level was significantly upregulated
212
in the kidneys, liver and spleen, while its expression levels in the gills were significantly
213
downregulated during the infection period. The expression levels of the PmCAPN3 transcript
214
in the kidneys and liver were significantly higher 3 dpi but decreased 5 dpi after the largest
215
increase occurred in the spleen 1 dpi. After RSIV challenge, significantly higher upregulation
216
was observed in the gills, kidneys and liver 3 dpi, but the expression level was downregulated
217
or maintained at the baseline level in the spleen. Tissue-specific expression analysis revealed
218
that the mRNA levels of PmCAPN3, a member of the cysteine protein family C2, were
219
significantly higher in the liver after pathogen infection than in other immune-related organs.
220
In previous reports, the cysteine proteinase family C1 (cathepsin B and L) was upregulated in
221
the liver following fungal and bacterial infections [26,27]. The liver plays an important role
222
in the innate immune response and is a key tissue for protein synthesis, which is essential for
223
protein homeostasis [28,29]. These results suggest that expression of the PmCAPN3
224
transcripts is induced and that the protein is activated mainly in the liver during pathogen
225
infection. CAPN3 was highly expressed in resistant animals [30]; however, our study also
226
showed significant increases in expression in immune-related tissues of P. major following
227
artificial infection with low concentrations of pathogens. The activity of CAPN protein is
228
increased in Leo virus-infected muscle cells, which is associated with apoptotic cell death 9
229
induced by viral diseases [31]. On the other hand, calpain-inhibited rats showed a significant
230
reduction in mortality after artificial infection with influenza A virus [32]. These results
231
suggest that it is important to control or understand CAPN signalling in diseases caused by
232
viral invasion. CAPN3 lacking protease activity was detected at high levels in human
233
melanoma cell lines, and CAPN3 is known to act as a regulator of existing CAPN systems
234
[33,34]. Furthermore, the CAPN3 gene is thought to be an important molecular marker in
235
aquaculture because it has been identified as a major muscle growth factor in breeding studies
236
of poultry and cattle [35–38]. Therefore, future studies on P. major breeding might suggest
237
that PmCAPN3 could be used as a candidate molecular marker. On the other hand, it was
238
significantly downregulated in the gills during nearly all periods of artificial infection by E.
239
piscicida (Fig. 4B). Previous studies have shown that E. tarda infects fish through the gills, a
240
major entry point [39] and organ vulnerable to infection by E. tarda. The infection is thought
241
to cause significant damage to the gills. Similar results confirmed that PmCAPN3 mRNA was
242
downregulated in the spleen, the organ most sensitive to RSIV infection (Fig. 4C). These
243
events might have disrupted the immune system in the tissues of hosts vulnerable to pathogen
244
invasion.
245
Our results also indicate that the PmCAPN3 mRNA expression level was upregulated by
246
pathogen infection via an in vivo analysis; however, this immunological result is not clear as
247
the protein level was not confirmed. CAPN3 is difficult to successfully purify because of its
248
strong and rapid autolysis, and full-length wild-type protein is a very unstable, making it
249
difficult to manipulate in vitro. Moreover, this protein shows rapid autolysis even in sodium-
250
containing solutions, such as buffered saline; thus, relevant studies are lacking, especially in
251
bony fish. This study identified significant upregulation of the PmCAPN3 transcript level
252
following pathogen invasion, confirming its potential as a pathogenic disease marker;
253
however, it also identified the need for further studies at the protein level. 10
254 255
Acknowledgements
256
This work was supported by a grant from the National Institute of Fisheries Science
257
(R2019058) and 'Smart Aquaculture Research Center', funded by the Ministry of Oceans and
258
Fisheries, Korea.
259 260
261
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Figure legends
384
Figure 1. Multiple alignment analysis of the deduced amino acid sequences of PmCAPN3
385
and other known CAPN sequences. This analysis is based on the following sequence data:
386
Atlantic halibut (ACY78226), turbot (AWP20177), Atlantic salmon (ACN10671), house
387
mouse (AAD28255) and human (AAD28253). The predicted catalytic domain and four EF-
388
hand calcium binding sites are indicated by the arrows and boxes, respectively. Black boxes:
389
identity = 100%; Grey boxes: 80% ≤ identity < 100%; Light grey boxes: 50% ≤ identity <
390
80%.
391
Figure 2. A phylogenetic tree based on the neighbour-joining method of PmCAPN and other
392
known CAPN homologues. The scale bar indicates a branch length of 0.2. The numbers show
393
the bootstrap percentiles from 1,000 replicates. The GenBank accession numbers are as
394
follows: Atlantic halibut CAPN3 (ACY78226), turbot CAPN3 (AWP20177), Atlantic salmon
395
CAPN3 (ACN10671), house mouse CAPN3 (AAD28255), human CAPN3 (AAD28253),
396
house mouse CAPN1 (AAH26138), house mouse CAPN2 (AAH54726), human CAPN5
397
(AAH18123), human CAPN6 (AAH00730), human CAPN7 (AAH56202), human CAPN8
398
(AAI57894), human CAPN9 (AAW49735), human CAPN10 (AAH07553), human CAPN11 16
399
(AAH33733), human CAPN12 (NP_653292), human CAPN13 (AAI17346), cattle CAPN14
400
(DAA24548) and human CAPN15 (NP_005623).
401
Figure 3. RT-qPCR-based analysis of the PmCAPN3 mRNA expression levels in various
402
tissue types in healthy Pagrus major. The PmEF-1α gene was used to normalise the RT-
403
qPCR results. The expression levels are reported as fold increases relative to the value in the
404
stomach. All data are presented as the mean ± SD from five independent cDNA samples with
405
three replicates per sample (N = 3).
406
Figure 4. RT-qPCR-based analysis of the PmCAPN3 mRNA levels in the gills, kidneys, liver
407
and spleen of Pagrus major after infection with (A) Streptococcus iniae, (B) Edwardsiella
408
piscicida or (C) red sea bream iridovirus (RSIV). The PmCAPN3 levels were quantified
409
relative to that of the PmEF-1α gene. The gene expression levels are represented as the
410
mean ± SD (N = 3). Asterisks indicate significant differences (*P < 0.05, **P < 0.01) versus
411
the control (0 h).
17
Catalytic domain Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Red sea bream Atlantic halibut Turbot Atlantic salmon Human House mouse
Figure 1. Hwang et al.
●
Figure 2. Hwang et al.
Figure 3. Hwang et al.
12
Relative expression level (Fold)
(A) S. iniae
**
10 8 6
**
**
4
*
*
* **
* *
2 *
0
Gill Relative expression level (Fold)
(B) E. piscicida
Kidney
Liver
Spleen
8
**
6
Cont. (0 h) 1h 12 h 1d 3d 5d 7d
** **
4
** *
*
2 ** ** ** ** **
*
*
0
Gill Relative expression level (Fold)
(C) RSIV
Kidney
Liver
25
Spleen
**
20 15 *
10
**
5
** ** *
*
0
Gill
Kidney
Liver
Figure 4. Hwang et al.
Spleen
Highlight The calpain 3 (CAPN3) was identified by NGS analysis from red sea bream (Pagrus major). PmCAPN3 showed high sequence similarity with other species CAPN3. PmCAPN3 mRNA was most abundantly distributed in the muscle and skin of healthy P. major. PmCAPN3 mRNA by fish pathogen challenge has showed a significant increase or decrease, and particularly showed the highest expression level in the liver.